Effect of Iron Deficiency in Offspring Rats: An Investigation of Fear and Anxiety-like Behavior Related to the Developing Hippocampus

Abstract

Introduktion: Jernmangel er en udbredt mikro ernæringsforstyrrelse og påvirker omkring 2 milliarder mennesker på verdens- plan. Behovet for jern forøges kraftigt under graviditet, fra 0.8 mg/dag i første trimester til > 6 mg/dag i tredje trimester, hvilket gør det svært at opretholde et tilstrækkeligt jern niveau uden brug af jerntilskud. Hippocampus spiller en vigtig rolle i bearbejdelsen af information og adfærd gennem en kompleks interaktion med amygdala og hypothalamus. Igennem oxidativ fosforylering bliver det høje energiforbrug fra hippocampus mødt. Cytochrome c oxidase er et jernholdig terminal enzyme som er involveret i sidste stadie af oxidativ fosforylering. Dette gør enzymet til en kvalificeret markør for neuronal metabolisk aktivitet. Formål: Formålet med studiet var at etablere et experimental model med gnaver for at undersøge jernmangel effekten på adfærd og den neurale udvikling i afkom fra rotter og hvorvidt tilskud med jern isomaltoside (Monofer) på to forskellige stadier (gestationsdag 1 og femte postnatale dag) af udviklingen kan forebygge hjerne abnormaliteter. Metode: 14 Wistar rotter blev inddelt i fire grupper; Kontrolgruppen (CN), gestationsdag 19 (G19), første postnatale dag (ID) og femte postnatale dag (P5). G19, ID, og P5 modtog alle jernmangel diæt og injektioner med Monofer på de tilsvarende dage. Afkommet blevet testet for frygt og angst-lignende adfærd med open field, elevated zero maze og light/dark boks, hvor udforskning var observeret i 10 minutter. Områder i hippocampus blev farvet for cytochrome c oxidase aktivitet og blev brugt til at understøtte observationerne i adfærdstestene. Resultater: I open field, viste stort set alle testene at CN var signifikant bedre sammenlignet med de tre andre grupperinger. Ved dag >28, viste G19 en forøgelse i distance sammenlignet med ID. Elevated zero maze viste en signifikant forøgelse i antallet af stræk mellem G19 og ID. I light/dark boksen viste ID og P5 sig at være mere signifikant end de resterende 2 grupper. Dot blot analyse viste ingen signifikant forskel mellem grupperne. Der blev dog observeret en tendens for CN, G19, og P5 til at have en forøget cytochrome c oxidase aktivitet sammenlignet med ID. Konklusion: Vægt og hæmoglobin niveau viste at jerntilskud havde en effekt sammenlignet med ID. Ad- færdsstudiet viste svagt at G19 klarede sig bedre sammenlignet med ID. P5 havde en tendens til at være bedre sammenlignet med ID. CN præsterede bedre i open field sammenlignet med ID og G19. Der var ingen signifikante oberservering i de biokemiske analyse.

Introduction: Iron deficiency is a widespread micro nutritional disorder and affects approximately 2 billion people worldwide. Iron demand increases notably during pregnancy, from 0.8 mg/day in the first trimester to >6 mg/day in the third trimester, thus, making it difficult to maintain an acquired iron level without any iron supplementation. Hippocampus plays an important role in processing information and behavior through a complex interaction with amygdala and hypothalamus. Through oxidative phosphorylation, the high energy requirement from hippocampus is met. Cytochrome c oxidase is an iron-containing terminal enzyme involved in the last step of the oxidative phosphorylation reaction. Thus, making it a quantifiable marker for neuronal metabolic activity. Aim: The aim of this study was to establish a rodent experimental model to investigate how iron deficiency will affect behaviour and neurodevelopment in offspring rats and whether supplementation with iron isomaltoside (Monofor) at two various stages (gestation day 19 and postnatal day five) of development can prevent brain abnormalities. Method: 14 Wistar rats were divided into four groups; Control group (CN), gestation day 19 (G19), postnatal day one (ID) and postnatal day five (P5). G19, ID and P5 received all iron deficient diet and injection of Monofer at the corresponding days. Offsprings were tested for any fear and anxiety-like behavior with open field, elevated zero maze, and light/dark box, where exploration were assessed for 10 min. Areas in hippocampus were stained for cytochrome c oxidase activity and used to support the findings in the behavior tests. Results: In open field, nearly all tests showed CN was significant different compared to all three groups. At day >28 in distanced travelled, G19 showed a significant increase compared to ID. Elevated zero maze revealed a significant increase in number of stretches between G19 and ID. Light/dark box showed that ID and P5 performed better compared to CN and G19. Dot blot assays did not reveal any significant difference between groups, however, there was seen a tendency for CN, G19, and P5 to have an increased cytochrome c oxidase activity compared to ID. Conclusion: Growth and hemoglobin showed that iron supplementation had an effect compared to ID. The behavioral study did not revealed any significant difference between the two supplementation groups compared to ID. However, CN had a better performance in open field compared to ID. There were no significant findings in the biochemical assays

A master thesis from Aalborg University

Effect of Iron Deficiency in Offspring Rats: An Investigation of Fear and Anxiety-like Behavior Related to the Developing Hippocampus

[Effekten af jernmangel i afkom fra rotter: En ungersøgelse frygt og angst relateret til den udviklende hippocampus]